Debenzylation of benzylated imidazolido-thiophane compounds



Patented Nov. 22, 1949 UNITED STATES PATENT OFFICE DEBENZYLATION OFBENZYLATED IMID- AZOLIDO-THIOPHANE COMPOUNDS No Drawing. ApplicationJune 8, 1948, Serial No. 31,846

9 Claims.

represented by the following formulae:

wherein R stands for hydrogen and benzyl, at least one R being benzyl; Ystands for an w-substituted alkyl radical, as for example, w-loweralkoXy-alkyl, w-halogen-alkyl, w-cyano-alkyl, wcarboxy-alkyl, andw-malonyl ester-alkyl [alkyl- CH(COOA1kyl)z], and Z stands for an anion,as for example, halogen.

The compounds represented by Formulae A and B, are new compounds and areintermediates in the production of biotin and homologs of biotin. Thecompounds and their mode of manufacture are described in our copendingapplications Serial No. 673,642, filed May 31, 1946, Serial No. 763,4 16and Serial No. 763,447, filed July 24, 1947.

In the aforesaid applications, the debenzylation of compounds of FormulaA is carried out with sodium and liquid ammonia.

According to the present invention, N-benzylated imidazolido-thiophanesin general, and of Formulae A and B in particular, can be readilydebenzylated with aqueous, preferably concentrated, solutions of halogenacids, such as hydrochloric, hydriodic, and hydrobromic acid.Debenzylation can also be carried out with anhydrous aluminum chloride.

The main products resulting from the new debenzylation method aredebenzylated 2-ketoimidazolido-thiophanes. Thus, the treatment of monoordibenzyl-biotin with the concentrated halogen acids, or aluminumchloride, gives biotin in good yield.

When the dibenzyl-(2-ketoimidazolido)-thiophanes are debenzylated, the

corresponding mono-benzylated, as well as completely debenzylatedcompounds, can be obtained. I-Iydrobromic acid is the preferreddebenzylating agent, since good yields are obtained and the reaction isreadily carried out. When a compound of Formula A, Y in the formulabeing an :0- malonyl ester-alkyl radical, as for example, 3,4-(1,3-dibenzyl-2-keto-imidazolido) 2 (w,wdicarbethoxy-butyl) -thiophane,is heated with concentrated hydrobromic acid, saponification,decarboxylation and debenzylation of this compound is achieved in oneoperation with the direct formation of biotin. This represents aconsiderable simplification of the process described in our applicationSerial No. 763,446 where three separate operations are required toobtain biotin from the above-mentioned dicarbethoxy compound.

In the debenzylation reaction with halogen acids, varying amounts of thedebenzylated 3,4- (2'-keto-imidazolido),-thiophane formed may be splitto the corresponding 3,4-diamino-thiophane, which can be easilyreconverted by reaction with phosgene to the said imidazolido-thiophane.For example, when 3,4-(1',3-dibenzyl-2'-ketoimidazolido) 2-(w,wdicarbethoxy butyl) -thio phane is converted by hydrobromic acid tobiotin, part of the biotin formed is split to give3,4-diamino-2-(w-carboxybutyl)-thiophane, which is easily converted tobiotin by treatment with phos gene in alkaline solution.

In general, the w-substituent in the side chain of compounds of FormulaA, when reactive to the halogen acids employed in the debenzylationreaction, can undergo secondary reactions. For example, if thew-substituent is an alkoxy radical, it can be converted into a halogenradical.

Our new debenzylation process provides better overall yields as comparedwith the debenzylation process in liquid ammonia with sodium. Thereactions are more readily carried out and simpler equipment isrequired. All of these advan tages result in considerable reduction incosts.

The following scheme will serve to represent the reactions involved inour new debenzylation can be observed during this reaction. Thebenprocess: zylbromide is removed by ether extraction and f3 f3 22 H1?lTlR R1 1 NR RI? 111R CH--CH OBI-+11 CH---(i3H (3H, oH- OHmOCZH. H,CH-(CH2)1OC3H5 H, oil-03,

S S l l Br CHr-OH:

f3 f3 i r E CH-('1H CH(IJH H, CH-(CHahCl on, oH-OH, s (v) Br our-- 11,.

mnx NHLHX 011 OH in. cit- H,); o 0 OH (v11) i r u (IJH-(IJ H (|JHCH CE:/CH-(CH2)4C0OH 1 CH2 JH-(CHz)lCOOH x R HN \NH RN NR H- tH H-+Hl COOCgHg;H, H(CHa)4.C.OOH on: CH'(CH2)iOH-COOC2H5 s (.VIII) (IX) R=CH:CH5 X=Cl,Br

The following examples will serve to illustrate our invention:

EXAMPLE 1 Debenzylation of 3,4-(N-monobenzyl 2'ketcimidaz0Zid0)-2-(w-ethomy butyl) thiophane (I) with hydrochloric acidA mixture of 1 gram of 3,4-(N-monobenzyl-2'- keto-imidazolido) -2-(w-ethoxy-butyl) -thiophane (I) 4 cc. of acetic and 10 ccof concentratedhy-'- drochlorie acid is heated in a closed tube for 24 hours to 105 C.The mixture is diluted with water, purified with activated charcoal,filtered and concentrated to a small volume. Ethanol and ether are addedcausing the precipitation of small clusters of needles. The product isfiltered off and. recrystallized several times from a mixture ofmethanol, acetone and ether, forming crystals meltingat 216-217" C. ofcompound (V).

'In this case, in addition to debenzylation, the

ethoxy group is replaced by a chlorine atom.

EXAMPLE 2' 'Debeneylation of 3,4 (1',3 dibenzyl 2 doctothe almostcolorless aqueous solution is concentratedin vacuo. Absolute alcohol isadded to the residue and the precipitated crystals are filtered offafter a few hours, M. P. 190-197" C. The debenzylated compound (VI) isrecrystallized repeatedly from methanol, forming finally prisms meltingat 203.5-204.5 C. It is a new compound and forms part of our invention.

EXAMPLE 3 Debenzylation of 3,4 (1',3' diben'ayl 2 Icetoimidazolido) -2(wethorypropyl) thiophane A mixture of 10 grams of compound (X) and cc.concentrated (48 percent) hydrobromic acid is stirred and refluxed forfive hours. The mixture is then diluted With waterand extracted withbenzene. The aqueous acid solution is treated with some activatedcharcoal, filtered and concentrated in vacuo. The residual syrup isdissolved in a hot mixture of methanol and ethanol. The reaction productprecipitates after a few hours. It is filtered off and recrystallizedfrom methanol, forming prisms melting at 203.5-2045" C.

In this debenzylation reaction there occurs also a replacement of theethoxy group by bromine, and the final reactionproduct is the cyclicthiophanium compound ('VI) 5 EXAMPLE 4 Debenzylation ofl-dibenzyZ-biotin (IV) A. 10 grams of crude l-dibenzyl-biotin isrefiuxed for 2 hours with 100 cc. of 48 per cent hydrobromic acid. Thecooled reaction mixture is extracted with benzene, to remove thebenzylbromide and some impurities, and is then concentrated in vacuo todryness. The residue is dissolved in boiling water, filtered and stirredtill the temperature falls to 50-60 C., then some chloroform is added todissolve all of the partly debenzylated material and the stirred mixtureis cooled to C. The precipitated product is filtered off. It isidentical in every respect with d-biotin. The chloroform solutioncontains dmonobenzyl-biotin, which can be debenzylated again bytreatment with hydrobromic acid. The aqueous liquors from the biotincrystallization contain some diamino acid (VII), which can bereconverted into d-biotin by concentrating the solution to dryness andtreating the residue with alkali and phosgener B. 4 grams of crudel-dibenzyl-biotin is heated on the steam bath for eight hours with 40cc. of 5557 per cent hydriodic acid. The mixture is concentrated invacuo, treated with hot water and extracted with chloroform. The aqueoussolution is concentrated in vacuo to a small volume and the precipitateformed is recrystallized from hot water with the addition of somechloroform. The aqueous layer yields d-biotin, while d-monobenzyl-biotinis isolated from the chloroform solution.

C. A mixture of 2.2 grams of crude l-dibenzylbiotin, 2.4 grams ofanhydrous aluminum chloride and 30 cc. of chlorobenzene is heated fortwo hours to 120 C. Water and some dilute hydrochloric acid are added,and the chlorobenzene layer is separated. The aqueous part is extractedwith chloroform to remove all nondebenzylated material, and is thenconcentrated in vacuo to a small volume. The solution is cooled to 0 C.for several hours and the precipitated product is filtered ofi. It is inevery respect identical with d-biotin.

EXAMPLE 5 Direct conversion of l-3,4-(1,3'-diben2yZ-2-ketoimidazolido)-2- (w,o-dicarbethoscy-butyl) thiophone (IX) to d-biotz'n 65 grams ofl-3,4-(1',3'-dibenzyl-2-keto-imidazolido) 2 -(w,w-dicarbethoxy-butyl)thiophane (IX) are heated with 800 cc. of 48 per cent aqueous technicalhydrobromic acid. A vigorous evolution of ethylbromide starts after theinside temperature has reached 90-100 C., and it is distilled off as itis formed. After 30 minutes the inside temperature rises to 125 C. andthe distillate consists of ethylbromide and a few cc. of hydrobromicacid. The distillation is continued for to 1 hour until 100-150 cc. ofhydrobromic acid, containing varying amounts of oily benzyl bromide,have been distilled off. The mixture is then refluxed another 2 /2 to 3hours for a total of 3 hours at 120-126 C. It is then concentrated todryness in vacuo (inside temperature 60 C.).

To the oily residue are added 1.8 liters of boiling water. The mixtureis boiled for minutes. The aqueous solution is then poured off from theoily, insoluble material which adheres to the flask. The solution iscooled down to 54 C., and the precipitated d-monobenzyl-biotin (prismsfrom dioxane melting at 181-182 C. corrected), is filtered ofi. It iscombined with the dark chloroformor dioxane-soluble material remainingin the flask, and is again debenzylated. To the aqueous solution areadded 200 cc. of chloroform and the mixture is stirred and cooled fortwo hours. It is then left for 24 hours at 0 C. The precipitated crudebiotin is filtered oif, the chloroform solution is separated and theaqueous solution is concentrated to a small volume and cooled to 0 C.yielding some more biotin. The d-biotln thus obtained is recrystallizedonce from 1790 cc. of boiling water with the addition of 1.0 gram ofactivated carbon and yields chemically pure d-biotin having a specificrotation of +92.'7 (2 per cent solution in 0.1 N NaOH) and a meltingpoint of 230-231 C. (corrected).

The aqueous mother liquor remaining after separation of the secondbiotin crop from the reaction medium contains some dihydrobromide of thediamino acid (VII) which can be reconverted to biotin by treatment ofphosgene in alkaline solution.

EXAMPLE 6 Debenzylation of d.l-monobenzyl-biotin A mixture of 1 gram ofd.l-monobenzyl-biotin (III), 4 cc. of acetic acid and 10 cc. ofconcentrated hydrochloric acid is heated for 24 hours to 108-115 C. in aclosed tube. The brown reaction mixture is diluted with water, activatedcharcoal is added and the mixture is filtered. The aqueous solution isextracted with ether, and concentrated in vacuo. To the crystallineresidue a small amount of methanol is added, followed by some ethanoland ether. The crystalline precipitate is filtered. It is recrystallizedseveral times from a mixture of methanol, actone and ether, formingneedles melting at 220-221 C. The compound obtained is thedihydrochloride of d,l-cis-3,4- diamino- 2 (w-carboxy-butyl) -thiophane(VII). In this case, debenzylation and splitting of the imidazolidonenucleus occurs. The compound can be easily converted to d.l-biotin byreacting it with phosgene in the presence of alkali, as for example, inthe following manner.

To a cooled solution of mg. of the diaminodihydrochloride (VII) and 0.8gram of sodium carbonate in 10-20 cc. of water, a xylene solution ofphosgene is added in small portions until the mixture becomes stronglyacid. The solution is then concentrated in vacuo and cooled to +5 C.After 20 hours, some precipitated impurities are filtered oil, and thesolution is cooled to +5 C.

i for 48 hours. d.l-biotin precipitates in clusters of needles and isfiltered off.

In general any of the N-benzylated imidazolidothiophanes of Formulae Aand B in optically active or inactive form can be debenzylated accordingto the invention in the manner herein described to form thecorresponding debenzylated imidazolido-thiophanes. The N-benzylatedimidazolido-thiophanes employed as starting materials can be prepared inthe following manner:

1,3-dibenzyl-imidazolidone- (2) -cz's-4,5-dicarboxylic acid and itsanhydride To a stirred, ice cooled solution of 648 grams (1.98 moles) ofbis-benzylaminosuccinic acid in 2 liters 3 N potassium hydroxide, areadded in small portions (within 1 hours) 1.6 liters of a 3.75 molarsolution of phosgene in xylene (=6 moles COClz) and 2.7 liters 6 Npotassium hydroxide (=14 moles). The mixture is then 7 acidified withconcentratedhydrochloric acid. and the formed: precipitate filtered offand washed .with water. The precipitate is then washed thor oughly withhot alcohol, and: the aqueous filtrate is extracted several times withethyl acetate. The alcoholic solution and the ethyl acetate extractcontain all the imidazolidone-dicarboxylic acid formed. The alcoholinsoluble part of the precipitate is pure starting material (220grams=0.65 moles). The alcoholic and: ethyl acetate solutions are takento dryness, and the residue is refluxed with benzene until it becomescompletely crystalline. The cooled mixture is filtered; the main part ofthe dibenzyl-imidazolidone-dicarboxylic acid remains on the funnel (283grams=0.8 moles). It can berecrystallized fromethyl acetate. Prisms,melting first at 167 C., thenresolidifying again and melting at 236 C,

The benzene motherliquor is concentrated in vacuo, and the rest of thereaction product isisolated in form of its anhydride in the followingway: The oily residue isrefiuxed with acetic anhydride, then the mixtureis concentrated and benzene is added. The anhydride formed crystallizesin nice needles. Melting point 23623'7 C.

Acetate of the cyclic form of 1,3-dibenzyl-cis-4- carbowy,formyl-zmidazolidone-2,3,4 (1,3- dibenzyl 2 keto-imidazolido)-2-keto-5-acetoxy-tetrahydrofumn A mixture of 100 grams of the anhydrideof 1,3 dibenzyl imidazolidone-cis-4,5-dicarboxylic acid, 150 grams zincdust, 100 grams zinc powder (40 mesh), 1500 cc. acetic anhydride and 500cc.

acetic acid is stirred and refluxed for -15 hours.

The dicarboxylic acidcan be used instead of the anhydride. In that casethe acid is first mixed with the acetic anhydride, refluxed for 10minutes and then the other constituents are added. The amounts of aceticacid and anhydride can be reduced to about /2 without affecting theyield. If too little is used, the yield is reduced. After that time, themixture is cooled, filtered and the precipitate on the funnel washedwith ethyl acetate. The filtrate is concentrated in vacuo, and the oilyor partly crystalline residue is treated with ice water and ethylacetate, until all the organic substance is dissolved. The ethyl acetatelayer is washed with water, dried with sodium sulfate and concentratedin vacuo. The oily or partly crystalline residue is boiled up withacetic anhydride. in order to reconvert any of the cornpound decomposedduring the treatment with water into the cyclic acetate. The mixture isagain concentrated in vacuo. Xylene is then added to the residue, andpart of it is distilled off in vacuo, then ether and petrol-ether areadded.

The reaction product separates in fine needles or prisms. which melt at103-104 C. After drying,

the melting point is l24-l25 C.

1,3-dibenzyZ-cis-4-carbomy-5-formylimidazolz'done-Z An excess of 3 Nsodium hydroxide solution is added to a solution of the acetate of thecyclic form of l,3-dibenzyl-cis-4-carboxy-5-formylimidazolidone-Z indioxane. After 2 minutes the mixture isacidified with dilute sulfuricacid and extracted with ether. The ether extract is washed, dried withsodium sulfate, and concentrated in vacuo. The residue is recrystallizedfrom a mixture of acetone, ether and petrol-ether.

The free aldehydo acid crystallizes in colorless needles or prismsmelting at 109-1l0 C. It forms a 2,4-dinitrophenylhydrazone, whichcrystallizes from a. mixture of dioxane, benzene and petrolether inorange prisms melting at 223.5-224.5 C.

The free aldehydo acid can be reconverted into the acetate of the cyclicform by treating it with acetic anhydride or acetyl chloride.

Thiolactone of 1,3-dibeneyl-cis-4-carboxy-5-mercaptomethylimz'dazolidone- 2 3,4-(1,3'-diibeneyl 2-keto-imidaeolido)-2-keto-thz'ophane A. A vigorous stream of hydrogen chloride andhydrogen sulfide is passed through a suspension of 150 grams of theacetate of the cyclic form of 1,3 dibenzylcis-4-carboxy-S-formyl-imidazolidone-2 in 850 cc. absolute alcoholcooled. to 10 to 20 C. After about 1% hours, most of the startingmaterial is" dissolved. To complete the reaction, hydrogen chloride andhydrogen sulfide are passed through the solution for another hour. Thesolution is then concentrated in vacuo, at the lowest possibletemperature and with exclusion of moisture. Some toluene is added to theresidue and distilled off in vacuo, thus removing most of the hydrogenchloride still present.

B. A vigorous stream of hydrogen chloride and hydrogen sulfide is passedthrough a solution of 150 grams of 1,3 dibenzyl-cis 4 carboxy 5-f-ormylimidazolidone-2 in 850 cc. absolute alcohol cooled to ID to -20C. After 2 hours the solution is concentrated in vacuo at the lowestpossible temperature and with exclusion of moisture. Some toluene isadded to the residue and distilled oil in vacuo, thus removing most ofthe hydrogen chloride still present.

C. The residual oil from reaction A or reaction B is dissolved in 1200cc. of a suspension of potassium hydrosulfide in alcohol. (Thissuspension is prepared by saturating a mixture of 672 grams potassiumhydroxide and 4 liters of alcohol with hydrogen sulfide). The mixture isleft at room temperature for 15 hours, then refluxed for one hour,poured on ice and acidified with dilute sulfuric acid. The organic partis extracted with ether, the solution is dried with sodiu sulfate andconcentrated in vacuo. The residue is reduced in a stirred refluxingmixture of 900 grams zinc dust, 150 grams granulated zinc (20 mesh) and2.3 liters acetic acid. After 4 hours, the solution is filtered, and themixture of zinc and zinc acetate remaining on the funnel is washed withether and water. The filtrate is concentrated in vacuo and diluted withether and 40 per cent sulfuric acid until clear separation takes place.The ether layer is separated, washed with water and concentrated invacuo to a small volume. After 24-48 hours, the mixture becomescrystalline. It is then diluted with some more ether and filtered.Melting point 123234 C.

The thiolactone can be recrystallized from a mixture of acetone, etherand petrol-ether. The pure compound forms colorless plates melting atl26-127 C. It is soluble in strong alkali, and gives a positivemercaptan test with sodium nitroprusside.

3,4- (1 ,3-dibenzyl-2 -lceto-imidazolido -2-hydroxy-Z- (w-ethomypropyl)-thiophane A Grignard solution, prepared from 13.6 cc. of3-ethoxypropyl-bromide and an excess of magnesium (4.8 grams) in 30 cc.of ether and 10 cc. of benzene, is diluted. with benzene, decanted fromthe unreacted magnesium and added dropwise (in about 30 minutes) to aboiling, stirred solution of 2'7 grams of3,4-(1',3'-dibenzyl-2-keto-imidazo- 1ido)-2ketothiophane (thiolactone)in 350 cc. of.

benzene. The solution is refluxed for another 3 hours. The mixture isthen decomposed with ice and dilute sulfuric acid, the organic layer isseparated and concentrated in vacuo. The residue is dissolved inmethanol and heated to 50 C. with an excess of aqueous sodium hydroxidesolution. Ether and water are added, and the two layers are separated.The alkaline aqueous solution contains the unreacted thiolactone, whichis extracted and recovered after acidification. The ether layer,containing the reaction product, is dried and concentrated in vacuo. Theresidue is crystallized from ether and petrol-ether.

The product is soluble in strong alkali and gives a positive mercaptantest with sodium nitroprusside. It can be recrystallized from a mixtureof acetone, ether and petrol-ether. Prisms melting at 114.5-115.5C. areobtained.

3,4- (1 ',3'-dz'benzyl-2-keto-imidazolido) -2- (w-ethoscy-propylidene)-thiophane A solution of 20.0 grams of 3,4-(1,3-dibenzyl- 2-ketoimidazolido)-2 hydroxy-2-(w-ethoxypropyDthiophane in 100 cc. acetic acidis refluxed for 1 hours. The solution is concentrated in vacuo, theresidue is dissolved in ether and the solution is washed with dilutesodium carbonate solution. The ether solution is dried and concentratedin vacuo. The oily residue solidifies after a few hours. It can berecrystallized from petrol-ether and forms fine needles melting at62.5-63.5 0.

3,4- (1,3-dibenzyZ-2'-keto-imidazolido) 2- (w-ethomypropyl) -thz'ophaneThe crude 3,4-(1'3'-dibenzyl-2'-keto-immidazolido)2-(w-ethoxypropylidene) -thiophane (19 grams), obtained as describedabove, is dissolved in 150 cc. of methanol and hydrogenated in thepresence of 4 grams ofprehydrogenated palladium oxide at roomtemperature and atmospheric pressure. The calculated amount of hydrogenis taken up in about 30 hours. The catalyst is then filtered oil, andthe solution is concentrated in vacuo. The product solidifies after sometime and is used in the crude form for the next step.

3,4 (1 3-dibenzyl-2Jceto-imidazolido) -1,2-

trimethyZene-thiophanium bromide A solution of 5 grams of3,4-(1,3-dibenzyl-2'- keto-imidazolido) -2- (w-ethoxypropyl) -thiophanein 50 cc. of an 18 per cent solution of hydrogen bromide in acetic acidis heated for three hours to 60 C. The solution is concentrated invacuo, and treated With water and benzene. The mixture is cooled to +5C. and the precipitated crystalline reaction product is filtered off andwashed with benzene and cold water. A further amount of the product canbe obtained by concentrating the aqueous part of the mother liquors (thebenzene layer contains only impurities). The product can berecrystallized from water. Thick plates melting at 220-222 C. areobtained.

3,4-(1,3'-dibenzyZ-2-ket0-imidazolido) 2 (t dicarboicy-butyl) -thiophaneor (d,Z-deZta-carbowy-dzbenzyZ-biotin) 13.2 grams of dried3,4-(1',3'-dibenzy1-2'-ketoimidazolido) -1,2-trimethylene-thiophaniumbromide are added with stirring to a solution of 1.38 grams of sodium in300 cc. freshly distilled diethyl malonate. The reaction mixture isstirred at 140-150 C. for two hours. It is then cooled, and ethylacetate and water are added, whereupon the precipitated sodium bromidedissolves. The organic and aqueous layers are separated, and the latteris extracted twice more with ethyl acetate. After drying the combinedorganic extracts with sodium sulfate, the solvents are distilled off invacuo, first using a steam bath and then an oil bath at about C. Theoily orange colored residue, containing the diethyl ester ofd,l-delta-carboxy-dibenzyl-biotin, is refluxed for 8-10 hours with 250cc. of methanol, 80 cc. of water and cc. of 50 per cent potassiumhydroxide. The reaction mixture is concentrated in vacuo to about of itsoriginal volume, and then about 1500 cc. of water are added. A slightprecipitate is formed, which is extracted with ethyl acetate. This is aneutral by-product. The aqueous solution is acidified with hydrochloricacid, whereupon an oil precipitates which is extracted with ethylacetate. After drying over sodium sulfate and distilling the solvent offin vacuo, a solid residue is obtained. This isd,ldelta-carboxy-dibenzyl-biotin. It can be recrystallized from amixture of acetone, ether and petrol-ether, and melts then at 133-135 C.with decomposition (evolution of carbon dioxide).

Decarboztg Zation of d,l-deZta-carboacy-dibenzylbiotin tod,Z-dibenzyZ-biotin 12 grams of d,l-delta-carboxy-dibenzyl-biotin arerefluxed for 10 minutes in 100 cc. of o-dichlorobenzene. The solvent issteam distilled and the brown residue extracted with ethyl acetate. Theethyl acetate solution is dried and concentrated in vacuo. The oilyresidue, weighing about 11.8 grams is dissolved in boiling benzene, thesolution is treated with charcoal, and filtered. Ether and petrol-etherare then added to the filtrate. After standing for at least 24 hours,d,l-dibenzyl-biotin crystallizes out. The product, which is somewhatcolored, is filtered oil and washed with ether on the suction funnel,until the filtrate is colorless. M. P. 109-112 C. After severalcrystallizations from a mixture of acetone, ether and petrolether, themelting point is constant at 122-124 C.

Debenzylation of d,Z-dibeneyl-bzotin to (Z1- monobeneyl-biotz'n 4.3grams of crystalline d,l-dibenzyl-biotin are dissolved in 125 cc. ofwarm dry xylene, and placed in a 3-neck flask fitted with a mechanicalstirrer and gas inlet and outlet tubes. An acetone-dry ice bath isprovided. The solution is stirred and about 250 cc. of dry liquidammonia are introduced. The acetone bath is removed and 1.15 grams ofsodium are added in small portions during the course of five minutes.The blue color of the solution remains even when it is stirred foranother half hour. Ammonium chloride is added to destroy the excess ofsodium. After distilling off the ammonia, and adding water and ether,the reaction mixture is acidified to pH 1 with hydrochloric acid andcooled to about 5 C. The crystalline precipitate is filtered oil. Itmelts at -1'72 C. Further purification is obtained by recrystallizationfrom about 2 liters of boiling water. 0n standing, the productcrystallizes out. It melts now at -176 0. and shows no melting pointdepression when mixed with a known sample of pure d,l-monobenzyl biotin.The aqueous filtrate is extracted three times with chloroform, and thechloroform, after drying, is concentrated in vacuo. The residue consistslikewise of d,l-monobenzyl-biotin.

Z-3,4(1',3-dibenzyZ-2'- keto imidazolido) 1,2 trimethyZene-thiophaniumd-camphorsulfonate or (l-thiophanium d-camphorsulfonate) 36 grams ofsilver carbonate are treated with a solution of 58.1 grams ofd-camphorsulfonie acid is 350 cc. distilled water. The solution ofsilver d-camphorsulfonate thus obtained is decanted from a small amountof undissolved silver carbonate, and is then added to a boiling solutionof 111.4 grams of 3,4-(1,3'-dibenzyl-2-ketoimidazolido)-1,2-trimethylene-thiophanium bromide in 3 liters of distilled water.The precipitated silver-bromide is filtered off, the solution isconcentrated in vacuo to dryness, and the dry residue is dissolved in2100 cc. of boiling isopropanol. After standing at room temperature for24 hours, the crystalline voluminous precipitate formed is filtered oil.The yield is 61.2 rams: 32 per cent of a product having a specificoptical rotation [a] of ---l.7i0.2. The material can be further purifiedby recrystallization from isopropanol. Needles melting at about 238-239"C. are obtained. The specific optical rotation of the pure material is[a] -2.6. The dextrorotatory antipode is contained in the motherliquids.

A mixture of absolute alcohol and ether can also be employed for theseparation Of the .land d-isomers. Thus 9.3 grams of the crude mixtureof the d-camphorsulfonates is dissolved in 50 cc. of absolute alcohol.'70 .cc. of ether are added, and the precipitated crystals are filteredafter one hour. The material thus obtained is, however, slightly lesspure; [0615 0. l.

Z-3,4- (1 ,3'-dibenzyl-2-keto-imida2olido) 2-.(w,w-dicarb0a:y-butyl)-thiophcme 43.5 grams of l -'3,4 -(1",3- dibenzyl 2- ketoimidazolido 1,2trimethylene thiophanium d-camphor sulfate -('[-u']D --1.0 to -2.6) and60 cc. of toluene are added to a stirred warm (70 C.) solution of 5.05grams of sodium in '80 grams of diethyl malonate. The mixture isrefluxed for three hours, it is then-cooled to room temperature, dilutedwith ether and washed with ice water to remove some dark impurities. Theorganic solution is concentrated .in vacuo, and the oily residue isdissolved in.200 cc. of methanol. 300 cc. of a 50 per cent aqueouspotassium hydroxide solution are added and the mixture is refluxed forfive hours. The cooled solution is then diluted with water, acidifiedwith concentrated hydrochloric acid and extracted with ethyl acetate.The ethyl acetateextract is concentrated in vacuo, and the crude oilyreaction product is used for the next step. The product can berecrystallized from .a mixture of acetone, ether and petrol-ether andforms prisms melting at 133-137 .C. with decomposition. [M -4. l. (c:1.2 per cent in 0.1 .N sodium hydroxide').

l- 3,4 -(1',3'-- dibenzyl 2 keto imz'dazolido)- (w-carboacybutyl)-thiophane or Z-dibenzyl-bz'otin 600 cc. of xylene are added to thecrude oily l-SA (1,3 dibenzyl 2-' keto imidazolido) 2 (w,w dicarboxybutyl) thiophane (33-34 grams) obtained asdescribed above. The mixtureis refluxed until the initially undissolved product is completelydecarboxylated, and the dibenzylbiotin formed is completely dissolved(-20 minutes). The solution is then heated for another 20 minutes,during which time about 200 cc. of the solvent is distilled off. Therest of the solvent is removed by vacuum distillation. The oily residue,representing crude dibenzyl-biotin, is used for the next step withoutfurther purification. It is very difficult to crystallize. The crudematerial has a negative specific rotation.

12 3,-4- (.1,,3' dibenzyl 2 keto .zmidacolido h 2- hydroxy 2-(w ethoxybutyl) th-z'ophane -.or '(in its open form) 1,3 dibenzyl :cis 4mercaptomethyl 2 (wethoxyvaleryl) imidazoZ-idone 2 A Grignard solution,prepared from 8.5 grams l-ethoxy-butylbromide and an excess of magnesium(2.4 grams) in 15 cc. of ether and 5 cc..of benzene is diluted 'withbenzene, decanted from the 'unreacted magnesium and added dropwise (inabout 30 minutes) .to a boiling, stirred solution of 13.5 grams of 3,4'(1',3' dibenzyl 2' keto imidazolido) 2 keto thiophane (thiolactone) in160 cc. of benzene. The solution is then kept refluxing .ior another 2hours. The mixture is decomposed with ice and dilute sulfuric acid, theorganic layer is separated and concentrated in vacuo. The residue isdissolved in methanol and heated to 50 C. with an excess of aqueoussodium hydroxidesolution. Ether and water are added, and the two layersare separated. The alkaline aqueous solution :contains the unreactedthiolactone, which is extracted and recovered after acidification. Theether layer containing the reaction product is dried, and concentratedin vacuo. The residue :is recrystallized from a mixture .ofiacetone,:ether, and petrol-ether, and forms thick plates "melting at l15-115.50.

3,4 (1',3' dibenzyl 2' keto imidazolido) 2 (w ethoxybutylz'denethiophane 3,4 (133 dibenzyl -.2' keto .z'mz'd-azolido) 2 (w cthomybutyl)thz'ophane The 3,4 1',3 dibenzyl 2 keto imidazolido) 2 (w-- ethoxy-butylidene) thiophane (20 grams), obtained in the previous example, isdissolved in cc. of methanol and hydrogenated in the presence of 20-40grams of Raney nickel catalyst at room temperature and atmosphericpressure. The calculated amount of hydrogen is taken up in 30 hours.Palladium catalysts, as for example, palladium on charcoal, palladium onbarium sulfate and palladium oxide, can also be used. The catalyst 'isfiltered ofi and the solution is concentrated in vacuo. Thehydrogenation product is used without .further purification for thefollowing reaction. It can be recrystallized from petrol-.ether and.forms thin plates melting at 84-85 C.

3,4 (N -monobenzy.Z-J2 -keto-imidazolidoi) -.2-

'(w-ethoxy'butyl) -thi0phane A solution containing 7.1 .grams of .3,4-(:1.,3'- dibenzyl 2'-:kelto .imidazolido) 2-'(w-ethoxybutyl)-thiophane in .20-30 .cc. xylene is added to 20-30 cc. liquid ammoniacooledin a.-Dry.Ice bath. To the stirred mixture is added "sodium -.(.inform of small pieces) until a persisting ;blue color :develops (about10.78 gram sodium is used)- The last excess of sodium is destroyed withammonium chloride, the ammonia is evaporated, and dilute sulfuric acidand ether are added. The mixture is stirred for a while and thenfiltered. The bulk of the reaction product remains on the funnel; asmaller part is dissolved in the ether layer of the filtrate. The latteris separated (if some product precipitates, ethyl-acetate is added),dried and concentrated in vacuo. The residue is recrystallized fromacetone-ether, together with the material obtained by filtration. Theproduct can also be recrystallized from a mixture of acetone, ether andpetrol-ether. Fine needles melting at 159- l60.5 C. are obtained.

We claim:

1. The process of debenzylating an N-benzylated3,4-(2-keto-imidazolido)-thiophane which comprises reacting saidcompound with a hologen compound of the group consisting of hydrochloricacid, hydriodic acid, hydrobromic acid and aluminum chloride.

2. The process of debenzylating an N-benzylated 3,4-(2-keto-imidazolido)-thiophane which comprises reacting said compound with a concentratedaqueous solution of hydrobromioacid.

3. The process of debenzylating an N-benzylated 3,4(2'-keto-imidazolido)-thiophane which comprises reacting said compound with a concentratedaqueous hydrochloric acid.

4. The process of debenzylating an N-benzylated3,4-(2-keto-imidazolido)-thiophane which comprises reacting saidcompound with a concentrated aqueous solution of hydriodic acid.

5. The process which comprises reacting 3,4- (1',3'-dibenzyl-2-ketoimidazolido) -2-(w,w-dicarbalkoxy-butyl) -thiophane with a concentratedaqueous solution of hydrobromic acid to form biotin.

6. The process which comprises reacting l-3,4- (1',3'-dibenzyl-2-ketoimidazolido) 2-(w,wdicarbethoxy-butyl) thiophane with a concentratedaqueous solution of hydrobromic acid to form dbiotin.

7. The process which comprises reacting 3,4- (1',3'-dibenzy1-2'-ketoimidazolido) 1,2-trimethylene-thiophanium bromide with a concentratedaqueous solution of hydrobromic acid to form 3,4- (2-keto-imidazolido)-1,2-trim.ethy1enethiophanium bromide.

8. The process which comprises reacting 3,4- (l',3-dibenzyl-2'-ketoimidazolido)-:2-(w-ethoxypropyDthiophane with a concentrated aqueoussolution of hydrobromic acid to form 3,4-(2- ketoimidazolido)-1-2-trimethylene thiopha nium bromide.

9. The compound 3,4-(2'-keto imidazolido)- 1,2-trimethylene-thiophaniumbromide.

MOSES WOLF GOLDBERG. LEO HENRYK STERNBACH.

No references cited.

Certificate of Correction Patent No. 2,489,238 November 22, 1949 MOSESWOLF GOLDBERG ET AL.

It is hereby certified that errors appear in the printed specificationof the above numbered patent requiring correction as follows:

Column 3, Formula (III), for that portion reading )3 d IIN NH HN NRcolumn 8, line 73, for ket thiophane read keto-th'iophane; column 9,lines 34 and 45, for 13-dibenzy1 read I'I-dibenzyl; column 11, line 34,after imidazolido insert a closing parenthesis; column 13, line 18, for"h0logen read halogen; column 14, line 23, for 1-2trimethylene read1,2-tr'imethylene; and that the said Letters Patent should be read withthese corrections therein that the same may conform to the record of thecase in the Patent Ofiice.

Signed and sealed this 28th day of March, A. D. 1950.

THOMAS F. MURPHY,

Assistant Commissioner of Patents.

